Studies to examine the role of GTPases, tyrosine kinases, and other signaling pathways in platelet function have been restricted because of the inability to perform genetic manipulations in platelets. A major part of this proposal is to develop mouse models in which these pathways can be examined through the tissue-specific deletion of signal transduction components in platelets. The proposal will focus on two specific components of the ras signaling pathway, rap 1b and c-raf-1, but will be applicable to any pathway. Rap 1b, which is more than 50% homologous with ras, has the intriguing property of reverting ras transformed cells and is therefore considered an inhibitor of ras-dependent pathways while c-raf-1 is a critical ras effector in the MAP kinase pathway. Both proteins are substrates for cAMP-dependent protein kinase and are thought to mediate the inhibitory effects of cAMP. The applicant proposes the development of models that will be used to examine the function of these two proteins in platelets and their role in the inhibition of platelets by cAMP. Mouse embryonic stem (ES) cells with homozygous knock outs for rap 1b or c-raf-1 will be prepared by homologous recombination. Platelets deficient in rap 1b or c-raf-1 will be prepared from these cells by either preparation of transgenic mice or in vitro maturation of ES cells to platelets through thrombopoietin-stimulated megakaryocytopoiesis. Platelet function will be tested to determine the effect on aggregation, secretion, agonist-stimulated MAP kinase activation, inhibition by cAMP, and phosphoprotein formation in response to cAMP.